CN100443119C - inhalation of nitric oxide - Google Patents

Abstract

Use of inhalable gaseous nitric oxide (NO) in combination with a cyclooxygenase inhibitor for the manufacture of a medicament for treating pulmonary vasoconstriction or airway constriction in a mammal, including man, in order to counteract a hypo- or non-response to treatment with gaseous nitric oxide or nitric oxide donor only and/or to counteract a rebound response in the case of withdrawal of treatment with gaseous nitric oxide or nitric oxide donor only said combination being use in a therapeutically effective amount to accomplish relaxation of said pulmonary vasoconstriction or airway constriction. A method and a pharmaceutical preparation for the treatment of pulmonary vasoconstriction or airway constriction while using the above-mentioned combination.

Description

inhalation of nitric oxide

field of invention

The invention belongs to the field of medicine and is used for treating pulmonary vasoconstriction or airway constriction of mammals, especially human beings. More specifically, the present invention is directed to the treatment of individuals who have no or minimal response to inhalation of nitric oxide, or those who experience a rebound response upon cessation of inhalation of nitric oxide.

Background of the invention

M，Frostell C，Arnberg H，Hedenstierna G.Eur.Respir.J.1993，第6卷，第177-180页；

 M，Frostell CG，Hedenstrom H，Hedenstierna Am.Rev.Respi r.Dis1993，第148卷，第1474-1478页)。因而，例如在EP560928，US5485827，5873359，和WO92/10228中公开了一氧化氮来治疗支气管痉挛和肺部血管收缩的用途。尽管如此，我们发现这种治疗的方法表现出很大的个体间相互差异和个体内在的差异。此外，尽管一氧化氮吸入(INO)可以作为一种有效的手段来治疗患有肺动脉高压的患者，大约1/3的患者对于INO表现出低反应或者没有反应。除此之外，当撤消INO的时候，我们可以观察到肺动脉高压和氧合作用的双重恶化，这被定义为反弹效应(rebound response)。由于间断吸入一氧化氮而引起威胁生命的血管动力学的不稳定和死亡也曾经被报道过。逐步减少NO的使用剂量将延长NO的治疗时间但是仍旧不能消除反弹效应。Nitric oxide soothes the blood vessels of the lungs, especially when the blood vessels of the lungs are constricted by various diseases, which are exemplified in the following description. Nitric oxide also relaxes airway smooth muscle (Belvisi MG, Stretton CD, Barnes PJ. Eur. J. Pharmacol. 1992, Vol. 210, pp. 221-222). Laboratory animal experiments and human experiments have shown that inhalation of gaseous nitric oxide can weaken the airway contraction caused by various factors (Dupuy PM, Shore SA, Drazen JM, Frostell C, Hill WA, Zapol WM.J.Clin.Invest.1992 , Vol. 90, pp. 421-428;

M, Frostell C, Arnberg H, Hedenstierna G. Eur. Respir. J. 1993, Vol. 6, pp. 177-180;

M, Frostell CG, Hedenstrom H, Hedenstierna Am. Rev. Respi r. Dis 1993, Vol. 148, pp. 1474-1478). Thus, the use of nitric oxide to treat bronchospasm and pulmonary vasoconstriction is disclosed, for example, in EP560928, US5485827, 5873359, and WO92/10228. Nonetheless, we found that this therapeutic approach exhibited large inter-individual and intra-individual variability. Furthermore, although inhaled nitric oxide (INO) can be used as an effective means of treatment in patients with pulmonary hypertension, approximately one-third of patients exhibit low or no response to INO. In addition, when INO is withdrawn, we can observe a double deterioration of pulmonary hypertension and oxygenation, which is defined as the rebound effect (rebound response). Life-threatening angiodynamic instability and death due to intermittent nitric oxide inhalation have also been reported. Gradually reducing the dose of NO will prolong the treatment time of NO but still cannot eliminate the rebound effect.

The mechanisms responsible for the low reactivity and rebound effects during NO use have not been fully elucidated. There is a hypothesis that inhaled NO inhibits NO synthesis in vivo through a negative feedback mechanism. In one study an animal model was developed using endotoxin injection for at least 3 hours to induce a rebound effect on intermittent inhaled NO. This study formed the basis of the present invention. In addition, the experimental results show that the rebound effect is not only due to the negative regulation of endogenous NO synthesis, but may be more important because of the enhanced activity of vasoconstrictor factor 1 (ET-1). Another observation reports a negative relationship between the response to INO and the degree of rebound. Therefore, the weaker the effect of INO, the stronger the rebound effect. Davidson and coworkers found similar associations between INO hyporesponsiveness, nonresponsiveness, and rebound in cases of neonatal dyspnea. (D.Davidson, MD, Pediatries.1999, Vol. 104(2), pp. 231-236)

In our model, endotoxin infusion resulted in a two-phase increase in pulmonary arterial blood pressure and a decrease in PaO2. Initially, 15 to 30 minutes after endotoxin infusion, pulmonary hypertension is severely intensified, accompanied by increased concentrations of vasoconstrictor thromboxane A2 (TXA2) or other cyclooxygenase (COX) products. Then, 2.5 hours after endotoxin infusion, a second episode of stable pulmonary hypertension and tissue hypoxia accompanied by an increase in ET-1 occurred.

Many studies have reported that INO cannot reverse pulmonary vasoconstriction induced by thromboxane analogues. (Welte M et al., Acta Physiol Scand., 1995, July, Vol. 154(3), pp. 395-405). Ikeda S, et al. (Ikeda S., Shirai M., Shimouchi A., Min KY., Ohsawa N., Ninomiya I., J. Physiology., 1999, February, Vol. 49(1), pp. 89-98 ) reported that INO in combination with intravenous cycloprostaglandin can produce enhanced vasodilatory effects.

In addition, specific substituted phenylalkenic acids and esters which can inhibit the symptoms of bronchotension induced by leukotrienes or arachidonic acid are known from patents US4536515 and US4537906, respectively.

Finally, Lippton H.L. et al. disclosed cycloplus Oxygenase products, such as PGI2, do not mediate the vasodilation response to isoprenephrine, bradykinin, and nitroglycerin located in the pulmonary vascular bed.

Based on the above, it is very surprising that a combination therapy effective in the treatment of pulmonary vasoconstriction or airway constriction can be obtained according to the present invention, which removes or at least broadly attenuates the above mentioned negative effects of INO.

Summary of the invention

It is therefore an object of the present invention to provide suitable compounds for the relief of respiratory or pulmonary vasculature in individuals under-responsive or non-responsive to treatment with gaseous NO or NO donors.

Another object of the present invention is to provide suitable compounds for counteracting the attenuated or even life-threatening effects of NO alone, preferably for counteracting or eliminating the rebound effects of intermittent NO inhalation.

Another object of the invention is to realize the use of a pure inhalable drug.

Other objects of the present invention should be apparent to those skilled in the art after reading the following description.

The above objects and others of the present invention, which can be appreciated by a person skilled in the art after reading the following description, are achieved in the uses, methods and pharmaceutical preparations defined by the appended claims.

More specifically, the present invention firstly provides a combination of inhalable NO and cyclooxygenase (COX) inhibitors for the manufacture of medicines for the treatment of pulmonary vasoconstriction or airway constriction in mammals, especially humans , to counteract the low or no response to treatment with gaseous NO and NO donors alone, and/or to counteract the rebound effect exhibited when treatment with gaseous NO and NO donors is withdrawn. This combined use should be achieved under the effective dose of treatment to achieve the above-mentioned counteracting effect.

Thus, according to the present invention, we have surprisingly found that the reduced palliative effects of exogenous NO, or even the life-threatening effects that occur when NO is intermittently inhaled, are at least partly due to cyclooxygenase products.

More specifically, we have found that when cyclooxygenase inhibitors are used in combination with NO, the palliative effect of NO is enhanced, or prolongs the palliative effect of NO, or reverses the reduced effect of NO alone, or reduces or even eliminates the Rebound effect during intermittent use of NO inhalation.

Detailed description of the invention

According to the present invention, the combination of gaseous NO and COX inhibitors can be used in the manufacture of medicaments for the treatment of all types of constriction challenges in the respiratory and pulmonary vessels.

These systolic diseases can be due to various causes such as: traumatic injury, pulmonary fat embolism, acidosis, adult respiratory distress syndrome, acute mountain sickness, acute pulmonary hypertension after cardiovascular and pulmonary surgery, persistent pulmonary arteries in newborns Hypertension, perinatal aspiration syndrome, hyaline membrane disease, acute pulmonary thromboembolism, acute pulmonary edema, heparin-protamine reaction, hypoxia, and bronchial asthma (eg, Status asthmaticus).

One aspect of the present invention involves the production of a medicament for the treatment of mammals that are completely insensitive or hyposensitive to inhaled gaseous NO alone. This aspect has very important clinical implications, because when treating different patient populations with NO inhalation, usually a large proportion of patients are completely insensitivity or hyposensitivity to NO inhalation.

Another aspect of the present invention is the manufacture of a medicament for the treatment of the rebound effect of intermittent NO inhalation.

A more preferred implementation of the invention produces a medicament for the treatment of symptoms of bronchoconstriction, such as bronchoconstriction complicated by bronchial asthma, especially acute bronchial asthma or conditional asthma.

According to the invention, NO is preferably applied in a gaseous, inhalable state. For example, inhalation of gaseous NO in therapy has great advantages over the use of non-gaseous NO donors because gaseous NO does not contain particles or droplets that need to be distributed and transported to the respiratory system. Gases have long pathways for free diffusion, pass obstacles (such as constricted airways) more easily than particles and liquid droplets, and can dissolve directly in tissues without causing bronchospasm of embedding. The very beneficial effect of NO gas on pulmonary blood vessels and airway smooth muscle groups can be observed soon after inhalation of gaseous NO, which makes NO a very useful first barrier against bronchospasm and subsequent pulmonary Vasoconstriction. Longer-acting formulations may be inhaled if desired.

However, according to another embodiment of the present invention, NO is used in the form of an NO donor, for example, the donor can be a compound that releases NO. Known compounds capable of releasing NO in the practice of the present invention are nitroso or nitrosyl compounds, such as S-nitroso-N-acetylpenicillamine, S-nitroso-L-cysteine and nitrosylguanidine, which are characterized by physiological The part of NO that is released or converted into NO by the compound under certain conditions. In other compounds, NO acts as a ligand of the transition metal complex, and can be released from the compound or converted into NO under certain physiological conditions, for example, nitroprusside, NO - ferredoxin, or NO-heme complexes. More suitable nitrogenous compounds are those metabolized by enzymes that are endogenous products of the respiratory or vascular system and that generate NO free radicals , for example, arginine, glycerol trinitrate, amyl nitrite, inorganic nitrite, azide and hydroxylamine. These classes of NO releasing compounds and methods of synthesizing them are well known in the art. Preferred NO donors are A compound that releases NO so that only blood vessels in the respiratory tract and lungs are affected.

The NO donors used in the present invention may be in the form of a powder (e.g., a finely divided solid, which may be provided neat or mixed with a biocompatible carrier powder, or the same or more additional therapeutic compounds) or in a liquid (e.g., Dissolved or suspended in a biocompatible liquid carrier, optionally mixed with one or more additional therapeutic compounds), and can be conveniently inhaled by the patient in the form of a spray (preferably comprising particles with a diameter of less than 10um or Liquid droplet). The powder or liquid carrier suitable for inhalation is generally used in traditional asthma inhalation therapy, so it is widely known in the art. The skilled person can know the appropriate optimal dosage range for inhalation according to routine procedures.

The cyclooxygenase inhibitors used in this experiment may be any compounds that are considered suitable for use in mammals, especially humans, and which can be conveniently administered. Experiments performed in connection with the present invention used non-selective COX-inhibitors. Blocking the COX-2 enzyme showed increased and prolonged INO potency. Blocking both COX-1 and COX-2 enzymes blunted the rebound effect. The use of non-selective COX inhibitors should therefore be effective, but the use of more selective COX inhibitors may provide more favorable efficacy.

Some examples of COX inhibitors that may be in accordance with the present invention are as follows: diclofenac; alclofenac; nabumetone; meloxicam; meclofenam; nimesulide; paracetamol; Felcoxib; Celecoxib; DuP 697 (5-bromo-2-(4-fluorophenyl)-3-[4-(methylsulfonyl)-phenyl]thiophene); GR 32191 (((IR- α(Z), 2β, 3β, 5α))-(+)-7-5(((1.1'-diphenyl)-4-yl)-methoxy)-3-hydroxyl-2-(1- piperidine)-cyclopentyl)-4-heptenoic acid); fluthamide (or cGP 28238); NS 398 (N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide (methansulfonamide ); L-745,337 (N-[6-[(2,4-difluorophenyl)sulfur]-2,3-dihydro-1-oxo-1H-inden-5-yl]methanesulfonamide) ; DFU((5,5-Dimethyl-3-(3-fluorophenyl)-4-(4-methanesulfonyl chloride)phenyl-2(5H)-furanone); HN-56249((3- 2,4-dichlorothiophene)-4-methanesulfonamide-benzenesulfonamide); JTE-522((4-(4-cyclohexyl-2-methylazol-5-yl)-2-fluorobenzenesulfon amide); aspirin; indomethacin; and ibuprofen.

Among these particular compounds, aspirin, indomethacin, and ibuprofen showed higher selectivity for COX-1 inhibition, while others (at least most) were more selective for COX-1 and COX- 2 inhibition exhibited greater selectivity or a similar degree of selectivity relative to COX-2 inhibition. Acid addition salts thereof such as hydrochlorides are also useful.

Agents used in accordance with the invention can be administered using commercially available respiratory devices. Compressed NO gas is commercially available, typically a mixture of 200-2000 ppm NO in pure N2 gas. The above-mentioned NO-N ₂ mixed gas can be input into the inhaled gas in an amount of 1-100000nmol/min, or can be mixed with air, oxygen or other suitable carrier gas or mixed gas, and the concentration of the mixed gas is generally 1-180ppm. For prolonged inhalation, a range of 1-40ppm is usually used, but 1-80ppm or 1-180ppm of gas can be used for short periods, when short-term strong effects can be obtained. Particularly preferred ranges in the last-mentioned case are 20-80 ppm (eg 40-80 ppm) or 40-180 ppm, respectively. For more details about NO inhalation, please refer to the prior art, eg EP560928B1, the technology disclosed in which is hereby incorporated by reference in the present invention.

The NO and cyclooxygenase inhibitors may be administered in any order, or they may be administered simultaneously, in the latter case, either from separate sources at the same time or together, or in a manner derived from the above-mentioned NO and Administered as a mixture of cyclooxygenase inhibitors.

Cyclooxygenase inhibitors can be administered in the same manner as NO, for example, by inhalation, or by common pharmaceutical administration routes. Among these routes, reference may be made to sublingual, buccal and rectal administration, epidermal administration, and subcutaneous, intramuscular, intravenous or intraperitoneal injection procedures. Preferably, however, the cyclooxygenase inhibitor is administered by inhalation. Thus, the cyclooxygenase inhibitor can be a powder (e.g., a finely divided solid, neat, or powdered with a biocompatible carrier, or mixed with one or more additional therapeutic compounds) or can be liquid (e.g., dissolved or suspended in a biocompatible liquid carrier, optionally mixed with one or more additional therapeutic compounds), but in aerosol form that is more conveniently inhaled by the patient (preferably comprising particles or droplets with a diameter below 10 μm). Liquid carriers and powders suitable as inhalation vehicles are commonly used in traditional inhalation therapy for asthma and are thus well known in the art.

The cyclooxygenase inhibitor is used in a therapeutically effective amount, which will be readily established by one skilled in the art and which will depend upon the type of compound employed and the route of administration, among other factors. As should be apparent to those skilled in the art, in this context and generally in the specification and claims, the term "therapeutic" includes prophylactic treatment as well as treatment of an established condition. Furthermore, "therapeutically effective" is used in its general sense in the art, as defined in the above-mentioned patent application EP 560928 B1, and generally in this particular case, when the cyclooxygenase inhibitor reverses the gaseous It is therapeutically effective when the negative effects caused by NO are eliminated. As a guide in this regard, it may be added that the compound diclofenac sodium is generally It is effective. This corresponds to a dosage of about 0.1-5 mg/kg body weight, eg 0.15-3 mg/kg body weight when used in aerosol form. However, sometimes lower doses are effective, and other times higher doses are needed. For other compounds, this can be used as a basis for establishing appropriate dosages.

According to a second aspect of the present invention, the present invention provides a method for treating pulmonary vasoconstriction or airway constriction in mammals, especially humans, with the aim of counteracting the patient's hyporesponsiveness or Unresponsiveness, and/or counteracting the rebound effect exhibited by withdrawn treatment patients when only gaseous NO and NO donors are used. The above method comprises administration of NO in the form of gaseous NO or an NO donor in combination with a cyclooxygenase inhibitor administered to a mammal in need of such treatment by an inhalation procedure. The above combinations should be used in therapeutically effective doses to achieve the above counteracting effects.

Concrete and preferred embodiments of the above method are referred to those specific and preferred embodiments which have been described in connection with the use according to the invention.

Finally, according to a third aspect of the present invention, there is provided a pharmaceutical formulation for the treatment of pulmonary vasoconstriction or airway constriction in mammals, especially humans, the purpose of which is to counteract the low or non-responsiveness of the patient to treatment with gaseous NO and NO donors alone , and/or to counteract the rebound effect exhibited by patients when treatment with only gaseous NO and NO donors is withdrawn. The present invention uses NO in the form of gaseous NO or an NO donor in combination with a cyclooxygenase inhibitor. The above-mentioned gaseous NO and the above-mentioned cyclooxygenase inhibitor should be in the therapeutically effective dose of the above-mentioned treatment to achieve the above-mentioned counteracting effect.

For specific and preferred embodiments of the formulations described above, reference is also made to those specific and preferred embodiments of the use according to the invention described above.

The invention will be illustrated by the following non-limiting examples.

Example

Experiments were carried out to study the relationship of the cyclooxygenase inhibitor - diclofenac sodium - to NO inhalation, and more specifically its effect in terminating the rebound effect of said inhalation.

Materials and methods

animal preparation

(ZOLETIL，Virbac实验室)(6mg/Kg)和xylzry(ROMPUN，B ayer AG，德国)(2.2mg/Kg)诱导麻醉。在诱导后，将一根套管插入耳部静脉并注射入阿片样物质(5μg/Kg)(FENTANYL，抗原医药有限公司，Roscrea，爱尔兰)。用帕乌龙(PAVULON，Organon Technika AB，

瑞典)(0.2μg/Kg)松弛肌肉。持续注入安眠药(chlomethiazole，HEMINEVRIN，Astra，瑞典)(400mg/hour)、帕乌龙(2mg/hour)和芬太尼(150μg/hour)以维持麻醉状态。如需要，重复静脉注射0.2-0.5mg芬太尼。当皮切口不会引起心跳和血压的任何变化时，麻醉的程度就足够了。加入预热(38℃)的等渗盐500ml/hour以供水合作用。在余下的研究中，将动物安置于仰卧状态(背侧横卧)。The Animal Research Ethics Committee of Uppsala University approved this study. Twenty-six Swedish pigs weighing 24-29 Kg were used for the study. Pigs were injected intramuscularly with the neuroleptic, azaperonum (STRESNIL, Janssen, Belgium), 40 mg, before transport from the farm. With atropine (0.04mg/Kg), thiacycline (tiletamin) / azole flurazide

(ZOLETIL, Virbac Laboratories) (6 mg/Kg) and xylzry (ROMPUN, Bayer AG, Germany) (2.2 mg/Kg) to induce anesthesia. After induction, a cannula was inserted into the ear vein and an opioid (5 μg/Kg) was injected (FENTANYL, Antigen Pharmaceuticals Ltd, Roscrea, Ireland). With Pa Oolong (PAVULON, Organon Technika AB,

Sweden) (0.2μg/Kg) relaxes muscles. Continuous infusion of sleeping pills (chlomethiazole, HEMINEVRIN, Astra, Sweden) (400mg/hour), Paoolone (2mg/hour) and fentanyl (150μg/hour) to maintain anesthesia. Repeat IV 0.2-0.5 mg fentanyl as needed. The level of anesthesia is sufficient when the skin incision does not cause any changes in heartbeat and blood pressure. Add pre-warmed (38°C) isotonic saline 500ml/hour for hydration. For the remainder of the study, animals were placed in the supine position (dorsal recumbency).

A tracheotomy was performed after anesthesia, and a cuffed tracheal tube (inner diameter 6 mm) was inserted. Mechanical ventilation was provided with a volumetric ventilator (Siemens 900c). Airway pressure and accurate ventilation are recorded with a transducer inside the ventilator. The respiratory rate was maintained at 20 breaths per minute, and the tidal volume was adjusted to maintain the CO2 (PetCO2) pressure at 36-41mm Hg (4.8-5.4kpa) during tidal ebb. The inspiratory time was 25%; at the end of inspiration a pause lasting 5% of the entire respiratory cycle was applied with a 5-cm _H2O exhalation pressure (PEEP). The fraction of inspired oxygen (FIO2) is 0.5.

Catheterization and Blood Measurements

A triple-lumen catheter (Swan Ganz no.7F) with a rubber tip was inserted into the pulmonary artery through the right external jugular vein to take blood samples and record pressure, and a large-diameter catheter was inserted into the opposite jugular vein for infusion, and place its head into the superior vena cava. The right carotid artery was cannulated for blood sampling and recording of arterial pressure.

Catheters connecting the arterial, central venous, and pulmonary arteries were connected to adapted pressure transducers (Sorenson Transpac transducers, Abbott Emergency Treatment Systems, Illinois, USA) and recorded with a Marquette 7010 monitor (Marquette Electronics, Wisconsin, USA). pressure. The mean arterial pressure (MAP), mean pulmonary artery pressure (MPAP), heart rate (HR), central venous pressure (CVP), pulmonary capillary wedge pressure (PCWP) and cardiac output (Qt) were recorded. Airway pressure was recorded with a ventilator. Vascular pressures were averaged over the entire respiratory cycle with a central thorax as the zero reference.

Qt was measured by thermodilution: 10 ml of ice-cooled isotonic saline was injected as a bolus and Qt was calculated (cardiac output computer Marquette 7010, Marquette Electronics, Wisconsin, USA). At least three injections were made for each measurement, and the average value was calculated. The injection is evenly distributed throughout the breathing cycle. The exact volume of exhalation is recorded with each measurement. Collect pooled venous and arterial blood samples for blood gas analysis (ABL 3, Radiometer, Copenhagen, Denmark), simultaneously collect oxygen saturation and hemoglobin concentration (OSM 3, Radiometer, Copenhagen, Denmark) and 5 ml of arterial blood, separate plasma For biochemical analysis (see below).

lung damage

Endotoxin was administered intravenously at 25 μg/Kg/hour for three consecutive hours to induce acute lung damage, and then 10 μg/Kg/hour of endotoxin was used to maintain lung damage for the remainder of the experimental period.

Experimental procedure

Thirty minutes after surgery, baseline hemodynamic measurements were taken, blood samples were taken, and blood was collected for subsequent biochemical analysis. Endotoxin (LPS, E. coli 0111:B4, Sigma Chemicals, St. Louis, MO, USA) was injected intravenously at 25 μg/Kg/hour for three hours to induce acute lung injury. Responses were measured at 30, 60, 120, and 150 minutes after the start of endotoxin infusion. The pigs were then divided into 3 groups: "cyclooxygenase (COX) blocked group", "non-blocked group", and "control group" to analyze possible INO-boosting effects of cyclooxygenase inhibitors.

1. In the cyclooxygenase blockade group, endotoxin was continuously infused for 3 hours before inhalation of 30 ppm INO was started. Before starting INO inhalation, saline solution (300 mg/kg) of diclofenac sodium (Sigma D6899), a non-selective cyclooxygenase inhibitor, was injected intravenously. After 30 min, before cessation of NO inhalation, hemodynamics and gas exchange were measured, and blood samples were taken for biochemical analysis. At 5, 10, 15, and 30 minutes after NO withdrawal, measurements were taken to detect rebound responses. NO inhalation was then performed for another 30 minutes (ie 4 hours after the start of endotoxin infusion), and NO was stopped again, and the results were measured at the same time points as before. The aim was to detect whether the response to INO remained constant or changed, and whether any rebound effects became stronger or occurred after the first.

2. In the non-blocking group, no cyclooxygenase inhibitor was added. Otherwise, other steps are the same as the cyclooxygenase block group. In this study, lung tissue samples were taken from 5 pigs via sternotomy at 4 occasions (before, during and after NO inhalation).

3. In the control group, endotoxin was also injected, but no INO or cyclooxygenase inhibitor was added. Control pigs were studied at baseline and at time points coinciding with the onset and end of the 2 INO effects, and at the end of the experiment, 5 hours after the start of endotoxin infusion.

The study period was 300 minutes (5 hours) after initiation of endotoxin infusion, and the total study time, including anesthesia, preparation and baseline measurements prior to endotoxin infusion, was approximately 7 hours.

NO administration and recording of NO in breath

1000ppm NO in N ₂ mixed with O ₂ /N ₂ mixture and connected to the low flow port of the ventilator. The inhaled gas is passed through a canister containing soda lime to absorb all the _NO2 . At the inhalation branch of the ventilation tube, the concentrations of inhaled NO and _NO2 were measured by chemiluminescence (9841NOx, Lear Siegler Measurement Controls Corporation, Englewood Columbia, USA). Set the pressure of inhaled NO at 30ppm and the inhaled _NO2 at less than 0.5ppm.

Expression of COX-1 and COX-2 was assessed by Western blotting

Rinse the lung tissue pieces with 4°C PBS. In 5 volumes of ice-cold 0.05M Tris buffer (pH 7.4), which contains 0.5mM phenylmethylsulfonyl fluoride to inhibit proteolysis, homogenate method (Ultra-Turrax, Jenke and Kunkel , IKA Labortechnik, Staufen, Germany) for total protein extraction of lung tissue. Supernatants were collected and stored at -80°C until analysis. The concentration of total protein in the supernatant was determined by the Lowry method. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to nitrocellulose membranes. The blot was blocked overnight at 4°C in 5% BSA in TBS solution, and then anti-COX-1 (1:2500, Cayman Chemical, MI, USA, Ca 160108) containing 1% BSA, COX-2 (1:1500, Cayman Chemical, MI, USA, Ca 160108) in TBS solution and incubated overnight. Wash five times with TBS solution. Blots were incubated with horseradish peroxidase conjugated to goat anti-rabbit immunoglobulin G (IgG) (1 :2500 dilution, Vector Laboratories, Burlingame, USA). The blot was washed five times with TBS solution, and the antigen-antibody complexes were detected on photosensitive film with enhanced chemiluminescence reagents (Ameraham, Arlington Heights, IL, USA). All experiments were repeated three times, and the results of each experiment were analyzed using the program Image 1.6C of the National Institute of Health (NIH).

TXB2 plasma

Blood was collected in pre-cooled tubes filled with EDTA (10 mM, final concentration), and centrifuged (10 min, 40C). The concentration of TXB2 was determined with a commercially available enzyme-linked immunoassay kit (Thromboxane B2EIAkit Cayman Chemical, MI, USA, Ca 519031). Serum was collected from each tube and stored at -70°C until TXB2 concentration determination. To ensure that all samples are free of organic solvents, the serum is purified before adding it to the test wells. Purify TXB2 according to Cayman Chemical Introduction, first add 10,000cpm tritium-labeled TXB2 (3H-TXB2) to each sample, then add 2ml ethanol, shake at 4°C for 5 minutes, and then centrifuge at 1500g for 10 minutes, to remove precipitated protein. Collect the supernatant and mix it with ultrapure water. Pass the sample through a C-18 reverse phase column. Take 10% of the effluent for scintillation counting.

The ELISA was repeated twice and 50 μl of purified sample was mixed with 50 μl of tracer compound and 50 μl of antiserum on a microtiter plate. After 18 hours of incubation, 200 μl of Ellmans reagent was added to start the enzyme reaction, and after 30 minutes the absorbance at 405 nm of each vial was measured with a microplate photometer (Thermo Max, Molecular Devices). The concentration of TXB2 in the sample was estimated from a standard curve obtained by non-linear regression of the absorbance values of 8 known concentrations of TXB2 ranging from 7.5 to 1000 pg/ml. The coefficient of variation between and within tests was less than 10%. See TXB2Enzyme immunoassayKit 519031 Cayman Chemistry Handbook for more details.

Statistical Analysis

Means and standard deviations of the means were calculated for all variables under all study conditions. Analysis of variance (ANOVA) was used for comparisons for repeated measures; correction for multiple comparisons was performed. Differences at the P<0.05 level were considered significant.

result

Endotoxin-induced lung damage

Baseline levels of hemodynamics and arterial oxidation were identical to those measured in healthy pigs in previous experiments (Freden F et al., Br J Anaesth, 1996 Vol. 77(3), pp. 413-418). There were no differences between these three groups.

Endotoxin infusion caused a two-fold increase in MPAP in all groups 150 minutes after infusion. PaO2 was significantly reduced to half of the baseline level. In addition, heart rate increases and cardiac output decreases. In the UN-COX group, the measurements were not significantly different at 3 and 4 hours after the start of endotoxin infusion. In the control group, the measurements were not significantly different at 3 hours and 4 hours and 5 hours after the start of endotoxin infusion.

Inhalation and Termination of No

In the non-blocking group, inhalation of 30 ppm NO resulted in a 28% decrease in MPAP levels and a 54% increase in PaO2 levels 3 hours after the start of endotoxin infusion. After 30 min, when NO inhalation was terminated, MPAP levels increased rapidly (within 5 min) to 23% (9 mmHg) above pre-NO inhalation levels, resulting in a rebound effect. The PaO2 level decreased rapidly to the level before NO inhalation, but did not show a rebound phenomenon.

Four hours after endotoxin injection, when NO was repeatedly inhaled, the decrease in MPAP level and the increase in PaO2 level was very weak, and the change in PaO2 level was no longer significant compared with the experiment one hour before. Five minutes after the cessation of NO inhalation, the level of MPAP increased significantly again and was higher than the pre-NO level (+26%), and the PaO2 level dropped below the pre-NO level. Thus, a pronounced rebound hypoxemia can be observed.

There is an inverse relationship between the increase in PaO2 levels during NO inhalation and the magnitude of the rebound effect on cessation of NO inhalation. Thus, the weaker the response to inhaled NO, the greater the level of PaO2 drop when NO inhalation is terminated. No such relationship was observed for MPAP.

In the COX-blocker group, pre-treatment with difluorofenac reduced the level of MPAP. INO significantly decreased MPAP levels and increased PaO2 levels. However, neither MPAP nor PaO2 showed a rebound effect when INO was terminated. The degree of reduction in MPAP was not increased by the combined use of INO and diclofenac sodium. Nevertheless, the block group prolonged the duration of inhaled NO compared to the non-block group. Furthermore, in contrast to the weakened response to NO in the non-blocked group, INO increased MPAP and PaO2 by the same levels in the second NO inhalation trial as in the first one an hour earlier. No rebound effect was recorded again when INO was terminated. During endotoxin infusion, respiratory resistance increases. INO alone will not prevent or attenuate this increase. However, in the COX block group, the increase in resistance was significantly lower (P < 0.01), and resistance did not increase throughout the INO period.

In the control group, the levels of MPAP and PaO2 remained stable throughout the two hours, corresponding to the effect of INO in the experimental group. Therefore, the comparison between the INO group and the control group more clearly revealed the improvement of MPAP and PaO2 under 2 exposures to INO, the rebound effect of INO termination and the efficacy of COX inhibitors.

NO inhalation and cessation did not affect the expression of COX-1, whether it was 3 hours or 5 hours of INO experiment. In the non-blocked group, the expression of COX-2 was significantly increased 3 hours after endotoxin injection, and increased to almost 10 times the normal baseline level (p<0.01) at 5 hours. No matter in the 3-hour or 5-hour inhalation experiment, 30 minutes of INO did not affect the expression status. Likewise, the expression of COX-2 remained increased after the INO period.

Concentration of plasma TXE-2 factor

There were no significant differences in baseline levels among the 3 experimental groups. The level of plasma TEX-2 increased sharply (6000-8000pg/ml) half an hour after the start of endotoxin injection, and decreased to 2 times of the baseline 3 hours after injection, and this change was not observed in the three experimental groups. difference. In the non-blocked group, plasma TXE-2 levels did not change during NO inhalation, but increased at 5 min after NO reduction and peaked at 15 min after INO (p<0.05). Plasma TXE-2 levels continued to remain elevated (20000-40000 pg/ml) when the second INO experiment started 30 minutes later, and further increased again when the second INO was terminated. In the COX-blocked group, plasma TXE-2 levels did not increase during the first INO action and upon cessation. Plasma TXE-2 levels decreased during the second INO experiment and did not increase when INO was terminated.

Claims (13)

1. Use of inhalable nitric oxide in combination with cyclooxygenase inhibitors, wherein nitric oxide is gaseous nitric oxide or nitric oxide donor form, for production in the treatment of pulmonary vasoconstriction and respiratory tract in mammals Drugs capable of counteracting the rebound effect during withdrawal of treatment with gaseous nitric oxide or a nitric oxide donor alone during contraction, the combination of which can achieve the aforementioned counteracting effect in a therapeutically effective amount. 2. Use according to claim 1 for the treatment of humans. 3. The use according to claim 1, wherein said pulmonary vasoconstriction and airway constriction are associated with clinical symptoms selected from the group consisting of: traumatic injury, pulmonary fat embolism, acidosis, adult respiratory distress syndrome, acute mountain disease, acute pulmonary hypertension after cardiovascular and pulmonary surgery, persistent pulmonary hypertension of the newborn, perinatal aspiration syndrome, hyaline membrane disease, acute pulmonary thromboembolism, acute pulmonary edema, heparin-protamine reaction, hypoxia and Bronchial Asthma. 4. Use according to claim 3, wherein said airway constriction is associated with bronchial asthma. 5. Use according to claim 4, wherein said airway constriction is associated with acute bronchial asthma and status asthmaticus. 6. The use according to claim 1, wherein said medicament is an inhalable medicament. 7. The use according to claim 1, wherein said production involves a medicament suitable for administering said nitric oxide and said COX inhibitor in any order. 8. The use according to claim 1, wherein said pharmaceutical form is a composition comprising said nitric oxide and a cyclooxygenase inhibitor to achieve simultaneous administration thereof. 9. The use according to claim 1, wherein said cyclooxygenase inhibitor is selected from the group consisting of: sodium diclofenac; aceclofenac; nabumetone; meloxicam; meclofenam; Sully; Paracetamol; Rofecoxib; Celecoxib; DuP 697; GR 32191; Fluxamide; NS398; L-745,337; DFU; HN-56249; JTE-552; Aspirin; and ibuprofen; or an acid addition salt thereof. 10. The use according to claim 1, wherein the concentration of inhaled nitric oxide present in the carrier gas or gas mixture should be in the range of 1-180 ppm. 11. Use according to claim 10, wherein the concentration of nitric oxide is between 1 and 80 ppm. 12. Use according to claim 10, wherein the concentration of nitric oxide is between 1 and 40 ppm. 13. The use according to claim 1, wherein said nitric oxide donor is selected from the group consisting of: S-nitroso-N-acetylpenicillamine, S-nitroso-L-cysteine, nitroprusside , nitrosoguanidine, glyceryl trinitrate, amyl nitrite, inorganic nitrite, azide and hydroxylamine.